The vertebrate immune system has evolved to recognize non-methylated CpG-motifs in bacterial or viral DMA. Microbial DMA, as well as synthetic oligonucleotides based on these motifs, activate Toll-like receptor 9, which leads to the induction of innate immune responses. Recent evidence suggests that TLR9 is also activated by immune complexes from experimental animals and patients with autoimmune diseases such as systemic lupus erythematosus. Hence, TLR9 activation is likely to be an important cause of the inflammation associated with rheumatologic disorders. TLR9 is expressed in the endoplasmic reticulum (ER) of unstimulated cells. After DMA entry into cells, TLR9 moves to endosomal compartments where it binds DNA and signaling is initiated. The overall goal of this proposal is to understand the molecular determinants for TLR9/DNA interaction, as well as the molecular mechanism of receptor activation. Using a novel ligand binding assay, we will determine the influence of DNA size, structure and chemistry on binding to TLR9. Conversely, the effect of TLR9 glycosylation and structural components of the N-terminal domain will be characterized by testing TLR9 mutants for ligand binding. We will ascertain the influence of DNA binding on TLR9 aggregation by biochemical and fluorescence-based technologies (FRET, FLIM). The molecular basis for the subcellular distribution of TLR9 and for the trafficking of TLR9 to the ER will be explored by mutagenesis approaches and electron microscopy. To better understand the mechanism by which TLR9 contributes to chronic inflammation, cells from patients with SLE will be stimulated with CpG-DNA and analyzed for cytokine production by ELISA. Similarly, we will assess gene expression profiles of SLE cells by microarray. Confocal microscopy will be performed on cells from SLE patients to investigate the trafficking behavior of immune complexes and TLR9. Understanding the early stages of immune complex-mediated inflammation should ultimately lead to the development of better therapeutics for rheumatologic disorders.